This invention relates to short-term storage of cultured tissue. More particularly, the invention relates to short-term storage of cultured epithelial tissue sheets useful as skin wound dressings in a manner which maintains cell viability and colony-forming efficiency.
It has been a priority in the medical community to develop a skin wound dressing which will encourage new growth while preventing fluid loss and infection following skin wounds from burns, ulceration, or surgical excision. Bandages and dressings fail to protect large-scale wounds adequately, and various alternatives have been developed. Among these are split- and full-thickness grafts of cadaver skin, porcine skin, and human allografts and autografts. Most have proved unsatisfactory for covering large wounds since all but autografts eventually are rejected by the body in the absence of immunosuppressive therapy. Autografts are useful in small areas, but for massive injury, conventional autografting is not practical.
Green et al. have developed a method of culturing epithelial cell sheets several cells thick for repairing burns, ulcerations and other skin wounds. U.S. Pat. No. 4,016,036 discloses the method for serially culturing keratinocytes to produce stratified sheets of epithelium. U.S. Pat. No. 4,304,866 discloses the method of producing transplantable cell sheets by culturing keratinocytes and detaching the sheet from its anchorage substratum using an enzyme such as dispase U.S. Pat. No. 4,456,687 discloses agents useful to promote growth of epithelial cells. The disclosure of these patents are incorporated herein by reference. In the culture system developed by Green et al., epithelial cells divide rapidly on the surface of tissue culture dishes or flasks, and ultimately form a confluent, modestly stratified sheet of tightly interconnected cells. These confluent cultures can be released as a cohesive cell sheet by treatment, for example, with the enzyme dispase (see U.S. Pat. No. 4,304,866). The cultured sheets then may be stapled to petrolatum impregnated gauze, or other non-adhesive backing, transported in culture medium to the operating room, and applied to the patient.
Autograft materials prepared by these methods are preferred for burn dressings, but require time to culture. While the autografts are being cultured, it is possible to maintain the wound with allograft material which is effective as a temporary wound dressing. Cultured epidermal allograft material promotes healing of chronic skin ulcers and split-thickness graft donor sites. Cultured epidermal autograft and allograft material made by the method of Green, et al. are now available from Biosurface Technology, Inc. of Cambridge, Mass. for commercial use and clinical trials.
A severe, very practical limitation on the use of cultured epithelial grafts is their limited shelf life. The viability and colony-forming efficiency of the cells in the sheets fall rapidly after they are removed from the anchorage substratum. The cell sheets are extraordinarily fragile. They are reproducibly able to resume growth and form a differentiated epithelium when applied to wounds for a maximum of about eight hours after dispase treatment. This limits the locations to which grafts can be shipped to those in proximity to a production facility. Expanding the availability requires either many production facilities throughout the world, or development of a method of lengthening the viability interval for the cultured sheets.
The art is replete with descriptions of various tissue preservation methods including cryopreservation, use of special cell media, and certain packaging techniques. Cryopreservation allows for long-term storage by freezing the material in the presence of a cryoprotective agent. This agent displaces the aqueous material in the cells and thereby prevents ice crystals from forming. Numerous disclosed protocols vary the nature or amount of cryoprotective agent, and/or the time course, or temperature of the freezing process in an attempt to retain cell viability after a freeze-thaw cycle. See, for example, U.S. Pat. No. 4,559,298, U.S. Pat. No. 4,688,387, and especially EP 0 296 475.
A second method of potentially lengthening the viable storage interval involves selection of the medium which surrounds the cells. For example, U.S. Pat. No. 4,681,839 discloses a system for preserving living tissue separated from its host organism by placing the tissue in a gas-permeable bag containing a "biscuit" which releases electrolytes, a buffering agent, a chemical energy source, high-energy phosphate compounds, metabolites, and sorptive material to remove toxic debris. Also, the patents of DeRoissart describe a method and apparatus for preserving living tissue in a nutrient fluid pressurized with a biochemically inert gas. See U.S. Pat. Nos. 3,607,646 and 3,772,153.
A third method of potentially maintaining viability involves the use of various types of containers such as the corneal storage system described in the Lindstrom et al U.S. Pat. No. 4,695,536, or the container for storing solid living tissue portions of U.S. Pat. No. 4,630,448.
Tissues stored at non-cryopreservation temperatures are commonly stored at 4.degree. C. See Rosenquist et al., "Short-Term Skin Preservation at 4.degree. C.: Skin Storage Configuration and Tissue-to-Volume Medium Ratio" 9(1) J.B.C.R. 52-54 (1988).
Storing tissue by means of cryopreservation is a complicated and expensive process. It is not currently a practical approach for transporting grafts from a production facility to an operating room. None of the other systems has been shown to extend the storage viability beyond very short periods, i.e., eight hours. See, for example, Pittelkow et al., 86 J. Invest. Dermatol. 4: 410-17, 413-14 (1986).
This invention seeks to provide a means for extending cultured epithelial graft viability, and to allow extension of the storage and transport time from production facility to the operating room so that life-saving graft materials may be transported long distances while maintaining and/or improving their ability to resume growth and serve as a living epithelial wound covering. The invention also seeks to accomplish these objectives while avoiding shipping the grafts in costly cryopreservation chambers.